PROJECT SUMMARY The continued rise of antibiotic resistance is a major threat to public health and has already resulted in a number of pathogens resistant to all available antibiotics. About 80% of antibiotics are derived from microorganisms that have used them against their competitors for millions of years. Yet, antibiotic resistance has remained at remarkably low levels in nature, in stark contrast to the dramatic increase in resistance among human pathogens. A critical gap in our knowledge is how naturally antibiotic- producing microorganisms have mitigated the rise of resistance in their competitors. Here we hypothesize that antibiotic producers have not only evolved antibiotics but also a strategy for wielding them sustainably. We propose that this strategy involves the use of antibiotic cocktails that act collectively to ward off resistance, increase potency, and target a greater breadth of organisms. Comparative genomics provides a means for uncovering this strategy by leveraging the abundance of recently-sequenced microbial genomes. In this project, we will develop and experimentally validate a novel bioinformatics technique to discover new candidate antibiotics and combinations of antibiotics with synergistic activities. The goals of this project are to both supplement our existing arsenal of antibiotics and shed light on sustainable ways for employing antibiotics in the clinic. This project is highly innovative because it will develop a completely new method for mining microbial genomes for natural products such as antibiotics, and it will demonstrate the advantages of considering antibiotics in a holistic context rather than in isolation. Furthermore, it may challenge the current clinical paradigm of antibiotic monotherapy by determining whether cocktails of antibiotics offer a strategic advantage. This project holds the promise of changing clinical practice by extending the useable lifetime of antibiotics, revealing new drug candidates for future research, and developing a novel technical capability with medical applications. These outcomes are crucial for public health at a time when the rise of antibiotic resistance shows no signs of abating and its costs continue to mount.